The ExbB protein together with the ExbD and TonB proteins is involved in energy-coupled transport across the outer membrane of Escherichia coli. To understand this unusual process it is required to determine the subcellular location of ExbB and its transmembrane arrangement. Using ExbB-beta-lactamase fusion proteins as reporters for a periplasmic versus a cytoplasmic location of the fusion sites, and accessibility of ExbB in spheroplasts and cell lysates to aminopeptidase K, trypsin, and proteinase K, we arrived at a model of ExbB topology in the cytoplasmic membrane. Starting with the N terminus in the periplasm ExbB contains three transmembrane segments (residues 16-39, 128-155, 162-194) a small periplasmic loop and two large portions in the cytoplasm. Two of the 18 fusion proteins studied, ExbB34-beta-lactamase and ExbB41-beta-lactamase, conferred a high ampicillin resistance. Protease experiments revealed a high respectively low percentage of the molecules in a reverse transmembrane orientation. Both proteins were lacking positive charges at the inner side of the cytoplasmic membrane which determine the orientation of transmembrane segments.